Influenza A viruses (IAV) are significant human pathogens causing yearly epidemics and occasional pandemics. Past pandemics have resulted in significant morbidity and mortality. The 1918 influenza pandemic was thought to have resulted in the death of at least 675,000 people in the U.S., and 40 million people worldwide. Pandemics in 1957 and 1968, while less severe, were also of major public health importance. A novel influenza A virus of swine origin became pandemic in 2009, causing the first pandemic in 41 years. In addition, annual epidemic influenza causes are also very significant resulting in approximately 36,000 deaths in the US annually. Role of sialic acid binding specificity of IAV Hemagglutinin (HA) in virulence and pathogenesis: IAV HA is responsible for binding sialic acid containing glycoproteins on cells to initiate infection. The specificity of HA binding to terminal sialic acid (SA) residues is dependent on the conformation of the bond to the penultimate carbohydrate. In general, avian-adapted IAVs have a binding preference for SA alpha2,3 Gal and human-adapted IAVs have a binding preference for SA alpha2,6 Gal. To understand how changing HA binding specificity affects IAV virulence in a mouse model, we studied the growth, virulence and pathogenicity of a number of recombinant influenza viruses expressing wild-type and mutant avian and human HA genes created using reverse genetics on the backbone of a contemporary low passage human H1N1 virus. These studies revealed that the 1918 IAV HA sequence likely contains virulence motifs outside of the receptor binding domain. The role of the ribonucleoprotein complex in host adaptation and pathogenesis of IAV: The proteins that make up the IAV ribonucleoprotein (RNP) complex - PB2, PB1, PA, and NP are likely to play a significant role in pathogenesis of IAV infection since a virus that can replicate to a high titer may be more pathogenic, and mutations in these genes are also very likely to be crucial for host switch events in IAV since replication must occur in the context of the host cellular milieu. In order to model RNP activity in different host cell environments, we have a developed a quantitative in vitro reporter assay as a surrogate for viral polymerase function. In this system, the RNP complex from a high path H5N1 virus demonstrated a much higher RNA polymerase activity than either the 1918 or the modern human IAV RNP, while a low path avian RNP complex showed almost no activity in human cells. Interestingly, there are very few coding differences between the RNP complexes of the low path avian virus, the H5N1 virus, and the 1918 virus. A related set of experiments is evaluating the significance of PB1 specifically in viral fitness. Genetic analyses of the previous 3 pandemics of the 20th Century have shown that at a minimum the HA and the PB1 segments have been derived from avian IAV strains. To address this question, we have assayed the function of representative avian, interpandemic, and pandemic PB1s isolated from 1940 to 1968 by using the in vitro reporter assay described above. Indeed, in these assays, the substitution of a pandemic virus-derived PB1 resulted in statistically significant increases in activity of the polymerase complex. The virulence of the 1918 influenza virus is still incompletely understood. While studies described above demonstrate that the 1918 HA encodes virulence determinants in the mouse model, a comparative pathogenicity study was undertaken between the 1918 influenza virus and an early classical swine H1N1 influenza virus, A/swine/Iowa/1931 in mice and ferrets. Interestingly, the swine virus was shown to be equally pathogenic in both mice and ferrets to the 1918 virus, suggesting that virulence determinants in the 1918 virus are preserved in this early classical swine virus. Epidemic or seasonal influenza is also a significant cause of illness and death. The H3N2 influenza viruses circulating in the 2004-2005 season caused more influenza illness and death than in a typical year. We evaluated the dominant strain that emerged in 2004 and were able to show that mutations in the polymerase genes likely contributed to the enhanced pathogenicity of this strain as evaluated in ferrets and in in vitro studies. Clinical protocol: Influenza in the Immunocompromised Host (#07-I-0229): Influenza viruses cause significant morbidity each year, and past epidemics and pandemics have caused significant mortality. The impact influenza has or could have on the increasing number of patients who are immunocompromised due to acute and chronic diseases as well as immunosuppressive therapies is largely unknown. Limited information is available about the innate immune response to influenza and how chronic illness and immunosuppression may affect the immune response to the virus. We have been evaluating patients with influenza by recording their symptoms, collecting their blood and nasal fluid specimens, and isolating their viruses. We are mapping each patient's infection by performing genomic analysis of the viral isolates collected during the patients'infection (including analysis of sequences known to confer resistance to antiviral medication), measuring cytokine levels in blood and nasal fluid, and measuring influenza-specific antibody responses. Correlation of these results with clinical symptoms and clinical outcomes is being evaluated. Elucidation of these responses and the genomic changes the virus may undergo during infection and treatment for influenza in immunocompromised patients may help us to better understand the impact influenza has on the overall health of these patients, as well as what strategies may perform better in protecting or treating these patients during the emergence of a new pandemic. We have identified viral clones in these patient samples with mutations conferring expanded antiviral resistance. Characterization of these viruses in both in vitro and in vivo models is in progress. With the emergence of the new pandemic in 2009, we were able to expand the current protocol to directly recruit immunocompetent patients with influenza into the protocol. We plan to study the natural history of patients with the pandemic in the upcoming influenza season. We developed a rapid and sensitive RT-PCR assay that allows influenza virus samples to be subtyped as 2009 pandemic H1N1 or seasonal H1N1.